Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
AJEBAK 54 (Pt. 2) 137-147 (1976) THE SURFACE MORPHOLOGY AND THE CELL CYCLE OF MASTOCYTOMA TARCET CELLS: NO APPARENT EFFECT ON CELL-MEDIATED KILLING by L. M. CHING, J. B. GAVIN*, I. MARBROOK AND M . SKINNER (From the Departments of Cell Biology and Pathology", University of Auckland, New Zealand.) {Accepted for publication Febrtiarif 19, 1976.) Summary. Mastocytoma cells (P-185) have been separated by velocity sedimentation into fractions which were highly enriched for eells at discrete stages of the cell cycle. By scamiing electron microscopy it was shown that the surface morphology of the majority of cells in each fraction was characteristic of that fraction. No diiference could be detected between i.solated fractions and unfractionated cells in their ability to be lysed by cytotoxic lymphocytes. INTRODUGTION. The main stages of a cell cycle are defined in relation to the time of DNA synthesis and mitotic division (Howard and Pole, 1953). Many other characteristics have been shown to vary with the stages of division. Gell cycledependent changes in the external surface are of particular interest in stitdies on cell interactions. These changes include gross alterations in the structure of the membrane (Scott and Garter, 1971), fluctuations in membrane potential (Sachs, Stambrook and Ebert, 1974). surface charge (Brent and Forrester, 1967) and variations in siuface morphology (Porter, Prescott and Frye, 1973). As immune reactions against cells involve antigens on the surface of the target cell, the variation of antigenic sites at different stages of the cell cycle are potentiaHy important in immnne cytolysis. Gyclic variations of the histocompatihility H2 antigen have been observed with cultured mastocytoma cells (Pasternak, VValmsley and Thomas, 1971) and in virus transformed mouse lymphoma cells (Gikes and Friberg, 1971). The H2 antigens were maximally expressed in early G] and minimally in S-phase. The sensitivity of cells to antibody-mediated cytolysis has been examined throughout the cell cycle (Shipley, 1971; Lerner, Oldstone and Gooper, 1971) but cell-mediated cytolysis has not been studied in such detail. 138 L. CHING. |. GAVIN, f. MARBROOK AND M. SKINNER This paper describes an investigation of the surface morphology and susceptibility to cell-mediated immune lysis of mastocytoma cells at various stages of the cell cycle. The technicjiie of velocity sedimentation {Miller and Phillips, 1969) was used to obtain fractions which were higlily enriched for cells at a given stage of the cell cycle. MATERIALS AND METHODS. Cell line. The mastocytoma P185 was u.scd throughoiil. The line was inaintainecl a.s an ascitic tiiiiiour in (DBA/2 x C.,H) F, Iiybnd mice by transferring lO-"' cells every 8-10 days. Five clays after cell transfer, proliferating mastocytoma cell popiilation.s were removed from the peritoneal cavity in 5 ml of pho.sphate budered saline (PBS) (pH 6 5 017M NaCl 00034M KCI, OOIM NaoHPO,. OOIM KfL-PO^). Pulse labelling of cells. Cells vi'ere labelled in vivo hy intra peritoneal injection of 2-5 nCi {-'Hl-thyniidine (specific activity 240 mCi/inmole) or 0-5 ^Ci ('*C)-thymidine (.specific activity 59 inCi/ niinole). Velocity sedimentation. The general methods described by Miller and Phillips (1969) were used with some modifications. All sedimentations were conducted at 4° in a cylindrical glass chamber (10-25 cm diameter) with a conical base connected by Tygon tubing to a peristaltic pump. Fifteen ml PBS, followed by 3 x 10" cells in 10 ml of 4% calf serum in PBS, then 400 ml of gradient (7-25? calf serum in PBS) were pumped tlirough the base into the c'hamber. The cells were allowed to sediment for 3 li at unit gravit>, then 10 ml fractions were collected with an automatic fraction collector (30 fractions/h). The ability of the cells to form colonies in agar wa.s used to indicate that there was no loss of viability during fractionation. Measurement of radioactivity. Cells were collected on glass fibre filters, which were then washed with distilled water to remove calf serum and v\ith 5% trichloroacetic acid to remove acid-soluble radioactivity. They were then dried and plac-ed in vials with 5 ml scintillation fluid (7-5 g 2,5-diphenyIoxazole and 0-25 g 1,4-bis [2-(5-phenyioxazolyl )]-benzene to 2-5 litres of toluene). The radioucti\ity wa.s measured by liquid seintillatinn counting. Scatminfi electron microscopy ( SEM). Cells were fixed at room temperature in 30 niin changes of 0-5%, I I , 2% pho.sphate bufi^ercd glutavaldehyde then washed and dehydrated iu increasing concentrations of tertiary butanol (Wheeler, Seelye and Gavin. 1975). Drops of the.se suspensions were placed on aluminium stubs, dried under vacuum, coated with carbon, then gold-palladium, and examined in the .scanning electron microscope (Stereoscan 2A, Cambridge Scientific Instruments Ltd.. England). Cell-mediated imj}titne killing. Cytotoxic lymphocytes, sensitized against DBA/2 histocoinpatibility antigens, were obtained from spleens of (DBA/2 x C-jH) Fj hybrid mice which had been irradiated (800 rads) and injected 5 days previously with 10« nucleated spleen cells from CBA mice. Spleen cell suspensions were prepared by teasing out the .spleen into medium (RPMI 1640 GIBCO). The cells were irrigated through a 20 gauge needle and clumps were allowed to settle into ealf serum. GELL GYGLE STAGES OF TARGET GELLS 139 Cell-mediated lysis was measured by the '>' Ci-chrnmate ielea.se assay (Biunner ct al.. 1970). Four .\ 10" nia.stocyt()ma cells; were labelled with 20-30 (iCi ^''Cr-chromate in 0-5 ml balanced salt solution for 30 min at 37°, then washed thoroughly to remove unbound chromate. Ten thousand labelled cells were added to the appropriate dilution of spleen cells in a total volume of 0-5 ml medium (RPMI 1640). Cells were centrifujied at 90 g and. after a 4 h incubation at 37° in an atmo.sphere of 5% CO^ in air, 1-5 inl of PBS \va.s added to each tube. The cells were thoroughly dispersed and then scdimentet! by centrifugation. One ml of tlu" supematant was removed and the radioactivity coimted in a Cannna Counter. Results were expressed as: counts 100 pereent chromium released = = counts in in supematant supematant X totat counts meorporated into the target cells RadiomttogTaphy. Cells suspended in calf serum were smeared on to mieroscope slides, then fixed in absolute ethanol (10 min) and 2% acetic acid (5 min). The slides were washed in distilled water, dried and coated with Kodak NTB-2 liquid emulsion. After 5 days at 4° the radioautographs were developed and the slides were stained with 5« Ciemsa stain (5 min), washed in phosphate buller. dried and scored for "labelled" cells. RESULTS. The separation of asynchronous nuistoctjtoma cells into fractions containing cells at discrete stages of division. Ma.stocytoma cells growing as an ascitic tnnionr were taken in the middle of the exponential phase of growth and fractionated according to thek cell volume, using velocity sedimentation. To verify that the cell fractions represented discrete stages of the cell cycle, the sedimentation rate of a stib-population of ceils, labelled with ''H-thymidinc, was followed thronghont one growth cvcle. A pnlse of ^H-Tdr was injected intraperitoneally into a series of mice and the tumour cells harvested at various times thereafter. The total amount of •'H-Tdr was incorporated into acid-precipitable radioactivity in less than 10 min ( < 0 0 2 of generation time), Immediately before removing the ttnnour cells from the peritoneal cavity, the population received a second pulse of '"Gthymidine to label the cells in S-phase at the time of removal. This gave a reference population so that both the absolute and the relative seditnentation rates of cells could be estimated. The sedimentation profiles of radioactive cells which had grown for various lengths of time are shown in Fig. 1. When tho tritium- and '^carbon-thyniidine were added simultaneously, the radioactive population sedimented at a mean rate of 8-5 mm/h. As the interval between the incorporation of the two radioactive isotopes increased, the tritium-labelled sub-population sedimented at a higher rate than the standard S-phase cells. After 4 h, the •'H-labelled cells sedimented as two populations, indicating tbat some of the cells had passed through G:- and mitosis and had reached G,. The cells in G, sedimented at a rate of 6-3 mm/h. B\ 7 h, all the •'•H-labelled cells had divided and they 140 L. GHING, |. GAVIN, ]. MARBROOK AND M . SKINNER SEDIMENTATION RATE (mm per h) 4 5 4-5 6 5 8 5 IO'5 12 5 6 5 85105135 f lOO 5 h it A S^ .''.J 100 A \ I h <3 6 h A ^ a \ \ Q' »_(»<» 100 2 h 7 ti 50 z / \ -..* k** 2 d 100 3 h O 8 h o 50 J/ 100 50 A iJ V .// /•' I J 9 h \ Fig. 1. Sedimentation profiles nf nulioacti^-cly-lahelk'd nuvstocytoina cells troni mice which were injected with -'H-Tdr. From zero to 9 h after the ''H-Tdr injection, the mice were injected with '^C-Tdr. The cells were harvested 15 min later and fractionated. The radioactivity in each fraction is expressed as a percent of the maximum. A ~ A ''H radioactivity. Q--Q '''C radioacti\'fty. CELL GYGLE STAGES OF TARGET GELLS 141 appeared as a single peak of radioactivity in the plot of sedimentation rates. The cells in Gi continued to grow until they had moved to the original sedimentation position of 8 5 mm/h at 9 h. The total generation time was therefore 9 h. To ensure that the radioactivity in each fraction reflected the distribution of radioactive cells, the sedimentation profile of radioactive cells was assayed using radioautography. The results in Fig. 2 indicate that there was a close correspondence between the amount of radioactivity in each fraction and the nnniber of labelled cells. 100 - Z c £ o IU z 50 o ae Q. Io o 6 10 14 le 22 FRACTION NUMBER Fig. 2. A comparison of the number of "labelled" cells and the amount of radioactivity in eaeh fraction. Ma.stocytoma cells were putse-lahelled with '^H-Tdr. har%este(l :md fractionated. An aliquot of each fraction was remo\ed for liquid scintillation counting, and the remainder of the fraction was prepared for riidioautojjraphy. The number of labelled cells detected by radioautography and the radioactivity in each fraction are expressed as a percent of the maximum. 0—0 ''H radioactivity. A--A labelled cells. Calculation of the duration of stages of the cell ctjcle. The data shown in Fig. 1 were analysed to calculate the length of each stage of the cell cycle. The area of the peak of radioactivity in cells at the Gi stage was expressed as a fraction of the total ''H-thymidine in the mastocytoma poptilation. In Fig. 3 the fraction of radioactivity in Gi is plotted against time. Between 2-5 and 3 h after S-phase cells were labelled, radioactivity started moving into the fractions containing Gi cells, indicating that these cells retjuire approx. 2 75 h to pass through the G^ and M stages. All radioactive cells had 142 L. GHING, J. GAVIN, ]. MABBROOK .AND M . S K I N N E B divided after 7 25 h (Fig. 2). This wt)tikl be the length of time required for cells labelled in early S to progress through S, G^ and mitosis. Thus, the 9 h cell cycle time of mastocytoma cells ct)nsists of an S-phase of 4-5 h, Gi phase of 1-75 h and combined G^ and mitotic phase of 2-75 h. The surface inoTphologtj of celh at different stages of cell cycle. The majority of cells from three fractions, which were known to be highly enriched for cells at particular stages of the cell cycle, showed consistent and distinct topography, although each contained a small proportion of atypical and intermediate forms. Fraction 7. (Sed. rate 5-7-6-9 mm/h.) Gells from this fraction, predominantl\- cells at the G, stage, were generally spherical in shape and had a slightly roughened surface with short bulbous or "blt'b-like" protrusions. I 0 • • — • 0-8 I 0-6 Q 0-4 < P^ 0 2 J I I L TIME (h) Fig. 3. The axDpearance of radioacti\ity in G, tells following a "pulse label" of the cells with ••'H-thyniidine. The amount of radioactivity in cells whicb have divided (G|) is expressed as a fraction of the total ''H-thyniidine incorporated. This fraction is plotted against the time after addition of isotope. The data is derived from the results of Fig. 1. Fraction 11. (Sed. rate 7-8-9 2 nim/h.) This fraction, highly enriched for cells in mid S-phase, contained many rounded cells with sinut)us, ribbon-like surface projections giving tliein a "ruffled" appearance. CELL CYCLE STAGES OF TARGET GELLS 143 u c ja — c n O « 144 L. GHING, |. GAVIN, ]. MARBROOK AND M. SKINNEB Fraction III. (Sed. rate > 9 2 mm/h.) Although these cells were predominantly Go or mitotic cells, the data in Fig. 1 indicated that a very small nnmber of S-phase cells were present. Most of the cells in this fraction had a few small blebs and many long fine microvilli extending from their surface. A few cells were considerably larger than the rest and had a relatively smooth surface. Plate 1 summarizes the relationship between these appearances, the sedimentation rates and the phases of the cell cycle. Sub-populations of mastoctjtoma eells as target cells. The stisceptibility of mastocytoma cells to cell-mediated lysis was investigated using the -^'Gr-chromate release method of Brunner et al. (1970). Gells from three fractions, described above, were labelled with •'"Gr-chromate. Eacb fraction was found to be labelled with '"Gr-chronmte to the same extent as the xtnfractionated cells. Tbe lysis of cells was carried out over a range of targetIymphoid cell ratios and the results are expressed in Fig. 4. The extent of lysis of the individual fractions was indistinguishable frotn the degree of lysis of unfractionated cells. The rate of lysis of all samples was identical (unpublished data). " . bO o 50 40 LU U 30 20 10 100 SPLEEN:TARSET CELL RATIO Fig. 4. Cell-mediated immune lysis of mastocytoma cells at dilteient stages of the cell cycle by .sensitized spleen cells. Alitiuots from each .sample were incubated with graded numbers of spleen celis and the •'•>'Cr released from dauiiifjed cells after 4 h was measured. Eac-h point represents the mean of duplicate assays. O - O Gi- 0 - 0 S. A - A G., ^ M. • - • Non-fractionated ct-Ils. CELL CYCLE STAGES OF TARGET CELLS 145 DISCUSSION. By investigating the progress of mastocytoma cells through the cell cycle, it has been possible to isolate fractions highly enriched for cells at particular stages for morphological and target cell studies. Ovu- techni(iue of following a pulse-labelled population was based on the assumption that S-phase cells incorporate radioactive thymidine into acid-precipitable material at a similar rate clurinjT the whole S-phase. Two lines of evidence indicate tbat this assumption is valid. First, the sedimentation profile of cells which may be scored as "labelled" by radioautography is identical to the profile of radioactivity which was measured as total radioactivity per fraction (Fig. 2). The amount of radioactivity was therefore proportional to tbe number of labelled cells. Second, the radioactive cells behaved as a single population on fractionation. Miller and Pbillips (1969) have discussed the homogeneity of populations in their definition of intrinsic resolutions ^ (where SS is the width at half the height of the peak and S is the mean sedimentation position of the population from the origin). For a single homogeneous population, the intrinsic resolution limit has been reported as varying from 0-lS (Miller and Phillips, 1969) to 0-28 (Williams and Moore, 1973). The mean resolution of the total labelled sub-population in the profiles shown in Fig. 1 was 0-28. When the labelled cells sedimented as two sub-populations, those which had pas.sed mitosis and those which had yet to divide (Fig. 1, 5 h), the intrinsic resolution was 0-25 and 0-18 respectively. These values are in agreement with those of other workers and indicate that the -'H-labelled cells were homogeneous with respect to cell volume. Thus, the experiment described in Fig. 1 was essentially a means of following the change of volume of a synchronous sub-population of mastocytoma cells. The valnes we have obtained for the duration of the mastocytoma cell cycle from velocity sedimentation analysis are in good agreement witb the valnes obtained for cultured mastocytoma cells by Schindler et al. (1970) in which they used separate technicjues to determine the length of each phase. Longer values for the mastocytoma cell cycle were reported by Bergeron, Walmsley and Pasternak (1970) using a method which required colcemid to block cells in mitosis. However, the presence of colcemid bas been shown to slow down the progress of the cells (Williams aud Carpentieri, 1967). Data on the sedimentation rate of cells allowed the size of cells in individual fractions to be correlated with their stage in the cell cycle and showed tbat cells sedimeuting at 6-3, 8-5 and 9 9 mm/h were highly enriched for Ci, S, G2 + M cells respectively. Scanning electron microscopy demonstrated that mastocytoma cells show a wide variety of surface specialisations whicb are closely related to the stage in the cell cycle. The order of changes we propose (Plate 1) was based on the remarkable consistency with which tbe features were observed in fractions examined. Cell cycle related variation in the surface morphology of Chinese hamster ovarian (CHO) cells in culture has been reported by Porter et al. (1973). Their obsei"vatious differ from those in this report and this may be due to the different 146 L. CHING, J. GAVIN, ]. MARBROOK AND M. SKINNER modes of growth. Presumably our mastocytoma cells, growing free as an ascitic tumour, were not influenced by the constraints imposed on GHO cells which grew as monolayers attached to a surface. Recent reports have proposed that T lymphocytes can be distinf^uished from B lymphocytes on the basis of their tt)pography (Polliack et ai, 1973) but this conclusion has not been universally accepted (Thurman, Buur and Goldstein, 1975). Our observations suggest that cell surface morphology may be an unreliable criterion unless maintained throughout the cell cycle. Despite the marked differences in the appearance of the cell surface, the fractionated cells were indistinguishable from unfractionated cells in their susceptibility to cell-mediated immune lysis. It thus appears that the sensitivity of the mastocytoma cell-mediated lysis does not vary significantly during the cell cycle. This is interesting, as fluctuations in the expression of surface H2 antigens during the cell cycle have been reported in several cell lines. Cikes and Friberg (1971) noted that antigen concentration was highest during G] and lowest during S-phase. The sensitivity of cells to antibody-mediated lysis does correspond to cyelic fluctuations in antigen concentration in cultured mastocytoma cells (Pasternak et ai, 1971) and mouse lymphoma cells (Gikes and Friberg. 1971). These cells were most sensitive to anti-H2 sera in G, and then decreased in sensitivity during the S-phase as the antigen concentration decreased. In contrast, a human Iymphoid cell line was found not to vary in sensitivity to cytotoxic ajitibodies despite cyclical variations in aiitigen concentration (Pellegrino et ai, 1974), whereas YGAB murine tumour cells varied in sensitivit)' to antibody-mediated lysis when no difference in antigenic expression could be detected (Lerner et ai, 1971). There are probably many factors other than antigenic expression which influence the sensitivity of the eells to immune lysis. The present investigation suggests that there is no great variability in the sensitivity of mastocytoma cells to cell-mediated cytolysis during the cell cycle. It may be that the area of contact between the killer and target cell is such that only substantial differences in antigen concentration wonld contribute to differences in the efficiency of cell-mediated lysis. Acknowledgements. This work v\'a.s supported by the Medical Research Council of New Zealand, and in part by the Auckland Division, Caneer Society of New Zealand Inc. REFERENCES. BERGEBON, J. J. M., WALMSLEY, A. M. H., BRUNNER, K. T., MAUEL, J., RUDOLF, M., and PASTERNAK, C . A. (1970): *Fhospholipid synthesis and degradation during the Iife-cyele of P185Y mast cells synchronized with exeess of thyniidine.' Biochem. ].. 119, 489. and CHAPUIS, B . (1970): 'Studies of allograft immunity in mice. I. Induction, development and in vitro assay of cellular immunity.' Immunology, 18. 501. BRENT, T . P., and FORRESTER, J, A. (1967): 'Changes in surface charge of HeLa cells during the eell cycle.' Nature, Lond.. 215, 92. CKES, M . S., and FHIDEHG. S. (1971): 'Expression of H2 and Moioney leukemia virus transformed cell surface antigens in synchronized cultures of a mouse cell line.' Proc. nat. Acad. Sci., U.S.A., 68, 566. CELL CYCLE STAGES OF TARGET CELLS HowAHD. A., and PELC. S. R. (1953): 'Synthesis of dooxyriljomujleit.' acid in normal and irradiated cells and its relation to chromosome breakage.' Heredity, 6, 26L SA(:H.S. H. COOPER, N . R. (1971): 'Ceil cycledependent immune lysis of Moloney virus-transformed lymphocytes: presence of viral antigen, accessibility to antibody, and complement activation.' Proc. nat. Acad. Sci., U.S.A.. 68. 2584. MILLER, R. G., and PHILLIPS, R. A. (1969): 'Separation of cells by velocity sedimentation,' ;. Cell. Physiol. 73, 191. PASTKRNAK, C. A., WALMSLEY. A. M. H., and THOMAS. D . B. (1971): 'Structural alterations in the .surface membrane during the cell cycle.' /. Cell Biol.. 50. 562. Ei.LEcniNO. M. A., FERHONE. S.. COOPER, N. R., DiERicH. M. P.. and REISFELD, R. A. (1974): 'Variation in susceptibility of human lyniplioid cell line to immune lysis (luring the cell cycle.' ]. exp. Med., 140, 578. , A.. LAMPEN, N . . CLARKSON. B. D.. DE HARVEN. E . . BENTWICH, Z., ZEICEL, F . P.. and KUNKEL, 11. G. (1973): 'Identification of human B and T lymphocytes by scanning electron microscopy.' /. exp. Med.. 138. 607. (1973): 'Changes in surface morphology of Chinese hamster ovary cells during the cell cycle; /. Cell Biol., 57. 815. P. J.. ant! Expl Cell Res., 83, 362. R.. RAMHEIER, L., SCHAER, J. C. and GRIEDER. A. (1970): 'Studies on the division cycle of mammalian cells. HI. Preparation of synchronously dividing cell population by isotonic gradient centrifugation.' Expl Cell Res.. 59. 90. Scorr, R. E., and CARTER. R. L . (1971): "Structural changes in membranes of synchronized cells demonstrated by freezedeavajje.' Nature, New Biol., Lond.. 233, 219. SHIPLEY. W . U . (1971): 'Immune cytolysis in relation to the growth cycle of Chinese hamster celts.' Cancer Res.. 31, 925. , G, B., BAUR. P . S.. and GOLDSTEIN, A. L. (1975): 'Examination of lymphocyte membranes of athymic 'nude' mice by scanning electron micro.scopy.' Ann. N.Y. Acad. Sd.. 249, 155. WHEELER. E . E . . SEELYE. R. N . , and GAVIN. J. B. (1975): 'Freeze drying from tertiary butanol in the preparation of endocardium for scanning electron microscopy.' Stain Teehnol. (accepted for publication). WILLIAMS, J. P. G., and CARPENTIERI, U . (1967): 'Metaphase arresting compounds in embryos.' Nature, Lond.. 216. 613. WILLIAMS, PORTER. K.. PRESCOTT. D., and FHYE, J. STAMBROOK, tlHEHT. J. D. ( 1 9 7 4 ) : 'Changes in membrane potential (luring the ceil cycle.* SCHINDLEB, LEH.NEK. R. A.. OLDSTONE. M . B. A., and G.. 147 N . . and MCX)BE, M . (1973): 'Sedimentatiitn velocity characterisation of tiie cell cycle of granulocytic progenitor cells in monkey hemopoietic ti.ssues.' /. Cell. Physiol, 83, 81.